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Biocatalysis holds immense potential for pharmaceutical development as it enables synthetic routes to various chiral building blocks with unparalleled selectivity. Therein, solvent and water use account for a large contribution to the environmental impact of the reactions. In the spirit of Green Chemistry, a transition from traditional highly diluted aqueous systems to intensified non-aqueous media to overcome limitations (e.g., water shortages, recalcitrant wastewater treatments, and low substrate loadings) has been observed. Benefiting from the spectacular advances in various enzyme stabilization techniques, a plethora of biotransformations in non-conventional media have been established. Deep eutectic solvents (DESs) emerge as a sort of (potentially) greener non-aqueous medium with increasing use in biocatalysis. This review discusses the state-of-the-art of biotransformations in DESs with a focus on biocatalytic pathways for the synthesis of active pharmaceutical ingredients (APIs). Representative examples of different enzyme classes are discussed, together with a critical vision of the limitations and discussing prospects of using DESs for biocatalysis. [ABSTRACT FROM AUTHOR]

Biocatalysis can be applied in aqueous media and in different non-aqueous solutions (non-conventional media). Water is a safe solvent, yet many synthesis-wise interesting substrates cannot be dissolved in aqueous solutions, and thus low concentrations are often applied. Conversely, non-conventional media may enable higher substrate loadings but at the cost of using (fossil-based) organic solvents. This paper determines the CO2 production—expressed as kg CO2·kg product−1—of generic biotransformations in water and non-conventional media, assessing both the upstream and the downstream. The key to reaching a diminished environmental footprint is the type of wastewater treatment to be implemented. If the used chemicals enable a conventional (mild) wastewater treatment, the production of CO2 is limited. If other (pre)treatments for the wastewater are needed to eliminate hazardous chemicals and solvents, higher environmental impacts can be expected (based on CO2 production). Water media for biocatalysis are more sustainable during the upstream unit—the biocatalytic step—than non-conventional systems. However, processes with aqueous media often need to incorporate extractive solvents during the downstream processing. Both strategies result in comparable CO2 production if extractive solvents are recycled at least 1–2 times. Under these conditions, a generic industrial biotransformation at 100 g L−1 loading would produce 15–25 kg CO2·kg product−1 regardless of the applied media. [ABSTRACT FROM AUTHOR]

Biocatalysis will play a central role in future chemical conversions, aiming at more sustainable strategies while keeping efficiency and economics. The mere use of enzymes does not ensure the greenness of a process, and quantitative environmental metrics must substantiate this. Simple but meaningful approaches are needed, particularly when reactions are still at a preliminary phase of research. This paper explores the use of the E-factor and a gate-to-gate strategy to assess environmental metrics for biocatalysis at the proof-of-concept stage. The upstream part – the actual biocatalytic reaction – and the downstream part – the purification of the product – are considered, providing the complete E-factor of the synthesis. As a case study, the hydration of oleic acid catalyzed by oleate hydratases (OHY) is used. Depending on the reaction conditions (particularly the substrate loadings), and the chosen downstream, total E-factors range from 15–20 to 160, mostly comprising wastewater (from the reaction media) and solvent (from the downstream part). The evaluation is completed with the estimation of the Total Carbon Dioxide Release (TCR), kg of CO2 per kg of product, which provides comparable data to benchmark synthetic strategies. The timely identification of hot spots (large contributors to the environmental impact) enables researchers and practitioners to propose experiments to show that biocatalysis can be performed under more sustainable conditions. [ABSTRACT FROM AUTHOR]

Enzyme catalysis (biocatalysis) is a mature strategy to perform organic synthesis with high selectivity and under mild reaction conditions. However, despite their origin and biodegradability, the simple use of enzymes is not sufficient to validate a process as green or sustainable. To reinforce the value that biocatalysis may offer, enzymatic processes can be established in environmentally‐friendly media, such as water, neoteric solvents (e. g. deep‐eutectic‐solvents, DES), biogenic solvents (e. g. 2‐MeTHF or terpenes), or even solvent‐free systems (if substrates allow that). Furthermore, these options can be conducted in continuous biocatalytic processes, which are more efficient and sustainable. Hence, a triple synergy is generated, and very promising environmentally‐friendly options can be envisaged. This conceptual paper discusses these alternatives, providing relevant, recently reported, successful examples. [ABSTRACT FROM AUTHOR]

The oxidation of 5‐hydroxymethylfurfural (HMF) to 2,5‐diformylfuran (DFF) is a key reaction in valorizing biomass. DFF is hardly soluble in water, while HMF is often obtained from biorefineries in crude wet organic fractions. Thus, the reaction is challenging for both biocatalysis performed in aqueous media, and for chemocatalysis where the presence of water often results in catalyst poisoning. Galactose oxidase (GalOx) can selectively oxidize HMF to DFF and displays promising activity in aqueous‐organic media. In this study, GalOx was immobilized on ten carriers, assessing the immobilization yield, activity, and stability. Covalently immobilized GalOx catalyzed the oxidation of HMF to DFF in neat and water‐saturated EtOAc, and in biphasic systems of various water contents. At 50 % v/v H2O, the reaction was conducted at a semi‐preparative scale (50 mL) with no adverse effect on DFF yield. Some limitations arise, such as enzyme deactivation, and adsorption of DFF to the support, particularly in the aqueous fraction. Future options to upgrade the route may include designed stable enzymes under the presence of HMF/DFF, and the setup of microaqueous systems where DFF adsorption is minimized. The use of wet EtOAc media would be a promising approach in future biorefineries employing inexpensive crude wet organic fractions. [ABSTRACT FROM AUTHOR]

The use of deep eutectic solvent (DES)/water mixtures were explored for the selective enzymatic synthesis of α-monobenzoate glycerol (α-MBG) from glycerol and benzoic acid as substrates. Experiments were performed with four DES, three of them containing choline chloride (ChCl), combined with urea (URA), glycerol (GLY), and ethylene glycol (ETA) (in all cases ChCl/HBD 1:2 mol ratio), and another one formed with methylammonium chloride and glycerol (MA/GLY 1:3 mol ratio). The best conversions (99 %) were achieved with immobilized lipase B from Candida antarctica (CAL-B) when ChCl/GLY was used as the solvent and the substrate at the same time. The use of water as a cosolvent (8 % v/ v) led to a significant decrease in the viscosity of the DES, and full conversions were then reached. Reusability studies of the biocatalyst revealed a 37 % decrease in activity after the first batch, but the activity remained mostly constant for the rest of the cycles. [ABSTRACT FROM AUTHOR]

Deep-eutectic-solvents (DES) have emerged in the last decades as promising bio-based and biodegradable neoteric solvents for biocatalysis, with examples covering different enzymes (mostly hydrolases) and whole-cells (baker's yeast). This paper explores for the first time the use of benzaldehyde lyase (BAL), a thiamine-diphosphate dependent lyase (ThDP-lyase) able to catalyze the carboligation of aldehydes (CC bond formation) in different DES-buffer mixtures. By using choline chloride-glycerol DES, BAL remains fully active with excellent enantioselectivity at 60:40 DES-buffer (v/v), whereby a significant denaturation is observed at 70:30 mixtures. Remarkably, the use of choline chloride-urea DES as reaction media leads to full conversions with BAL at such solvent-buffer proportions, suggesting that the design of both the biocatalyst and the neoteric solvent may provide useful novel reactive systems for biocatalysis in non-conventional media. [ABSTRACT FROM AUTHOR]

Biorefineries are expected to take over the production of chemicals and biofuels in the coming decades, in parallel to the depletion of fossil resources. Considerable research efforts have been made to develop efficient (bio)catalytic systems for the production of bio-based solvents and novel products with improved ecological footprints and 'smart' properties. Among other challenges, water consumption will be an important aspect to assess in the full sustainability of biorefineries, which are envisaged for large-scale operations. The already existing fresh water shortage in many regions of our overpopulated planet has brought more pressure and uncertainties to these aims. Thus, over the past years, a number of enzymatic, fermentative, and chemocatalytic applications using seawater as a reaction medium for large-scale biorefineries have been reported by several research groups, which emphasize the use of nonpotable water resources for coastal regions and for locally available biomass (e.g., algae). This article contextualizes in detail these seawater-based applications for biorefineries. Overall, the use of such nonpotable water resources is promising, provided that applied catalytic systems can lead to straightforwardly treatable waste(sea)water effluents. Opportunities at the interface between biology, chemistry, and chemical engineering are foreseeable for (holistic) innovation and further research. [ABSTRACT FROM AUTHOR]

Abstract: This communication explores the use of seawater (1X) and concentrated seawater (2X and 4X) as reaction media for the enzyme-catalyzed depolymerization of cellulose. The commercially available Accellerase-1500® – a “cocktail” of different glycosidases – is able to depolymerize several amorphous celluloses and microcrystalline cellulose Avicel® in these reaction media, at slightly lower rates (ca. 90%) than those observed when reactions are performed in pure citrate buffer (control reactions). Remarkably, at concentrated seawater effluents enzymes also display significant rates of cellulose hydrolysis. Considering the expected increasing shortages in accessibility to fresh drinkable water, the herein-reported concept may provide novel inspiring leads for a smart use of resources in an environmentally-friendly and efficient manner, and for the genetic development of cellulases highly active and stable in concentrated seawater solutions. [Copyright &y& Elsevier]

The profen family belongs to the most important non-steroidal anti-inflammatory drugs (NSAIDs). A considerable number of biocatalytic processes for the synthesis of optically pure (S)-profens have been proposed. Despite of the excellent enantioselectivity and the large advantages that enzyme catalysis offers in terms of sustainability, biocatalytic processes have failed so far to meet the technical and economic challenges of commercialization. This critical review outlines recent trends and developments of novel applications that appear very promising in terms of enantioselectivity, efficiency, sustainability and yield. Special emphasis is placed on the contribution of protein engineering in overcoming the limitations of these enzymes for technical applications, and thus providing promising biocatalysts for the preparation of pharmaceutical products. The natural catalytic diversity, assisted by modern methods of protein engineering, provides novel concepts and leads for the environmentally friendly synthesis of pharmacologically important drugs. Considerable progress can be expected in the coming decades. Furthermore, aspects regarding ecological footprints and the impact of each biocatalytic route are critically addressed, considering aspects like the type of solvent, waste produced, availability of substrate, etc. When possible, suggestions for combining efficiency with more sustainable synthetic approaches are also given. [ABSTRACT FROM AUTHOR]

Ionic liquids (ILs) have been extensively assessed in biotransformations with different purposes, for example, non-conventional (co-)solvents, performance additives, coating agents for immobilizing/stabilizing enzymes, and IL-membrane-based processes. Fuelled by their premature labelling as ‘green solvents’, academic research has flourished. However, in recent years environmental aspects related to ILs have been strongly addressed, stating that many ILs commonly used cannot be regarded as ‘green derivatives’. Likewise, ILs costs are still a barrier for practical uses. Attempting to combine sustainability with the promising added-values of ILs, the third generation of ILs is currently under development. Likewise, deep-eutectic-solvents (DESs) appear in the horizon as an attractive and cost-effective option for using ionic solvents in biotransformations. DESs are often produced by gently warming and stirring two (bio-based and cheap) salts (e.g. choline chloride and urea). First successful uses of DES in biotransformations were reported recently. It may be expected that knowledge accumulated in (second generation) ILs and biotransformations could be turned into real applications by using these DESs, and third generation ILs, in the coming years. [ABSTRACT FROM AUTHOR]

Abstract: Lipase-catalyzed esterifications in biphasic media (heptane–water, 1:1) were conducted by using Thermomyces lanuginosus lipase (TLL) as biocatalyst. Different carboxylic acids (from acetic to lauric) were thus esterified with 1-butanol at different pH values (2–10). For all carboxylic acids tested, pH optima for the enzymatic esterifications were ca. 3–5, in clear agreement with previous literature, and quite different from optima pH of TLL in hydrolytic reactions (8–11). Interestingly, the interval of pH in which TLL was active in esterifications, varied markedly depending on the carboxylic acid. Thus, for long-chain acids (i.e. lauric), such pH interval was much wider than for short-chain carboxylic acids (i.e. acetic). To understand this effect, simple pK a values of carboxylic acids, retrieved from open literature, are not sufficient, since those values are measured in pure water. When a second phase is added, aspects related to partition coefficient of each carboxylic acid need to be considered as well, thus leading to the apparent pK a concept. Herein we performed theoretical calculations to obtain such app pK a of each carboxylic acid. When such calculations were compared with the pH interval, a clear correlation was observed. Overall, results confirm that lipases accept only the protonated form of the carboxylic acid (R–COOH) during esterifications in biphasic media. [Copyright &y& Elsevier]

Abstract: The use of oxidoreductases as biocatalysts for the production of a wide number of chiral building blocks is presently a mature (bio-)technology. In this context some industrial applications are currently performed by means of those enzymatic approaches, and new examples are expected to be realized. Moreover, oxidoreductases provide an interesting academic platform to undertake fundamental research in enzymology, to acquire a better understanding on catalytic mechanisms, and to facilitate the development of new biocatalytic applications. Within this area, a wide number of oxidoreductases from genus Candida spp. have been characterized and used as biocatalysts. These enzymes are rather diverse, and are able to carry out many useful reactions, like highly (enantio)selective keto-reductions, (de)racemizations and stereoinversions, and promiscuous catalytic imine reductions. In addition, some Candida spp. dehydrogenases are very useful for regenerating the cofactors, with the aid of sacrificial substrates. Addressing those features, the present paper aims to give an overview of these enzymes, by focusing on practical applications that these biocatalysts can provide. Furthermore, when possible, academic insights on the enzymatic performances will be discussed as well. [Copyright &y& Elsevier]

Abstract: The enzymatic promiscuity concept involves the possibility that one active site of an enzyme can catalyse several different chemical transformations. A rational understanding of the mechanistic reasons for this catalytic performance could lead to new practical applications. The capability of certain hydrolases to perform the perhydrolysis was described more than a decade ago, and recently its molecular basis has been elucidated. Remarkably, a similarity between perhydrolases (cofactor-free haloperoxidases) and serine hydrolases was found, with both groups of enzymes sharing a common catalytic triad, which suggests an evolution from a common ancestor. On the other hand, several biotechnological applications derived from the capability of hydrolases to catalyse the synthesis of peracids have been reported: the use of hydrolases as bleaching agents via in situ generation of peracids; (self)-epoxidation of unsaturated fatty acids, olefins, or plant oils, via Prileshajev epoxidation; Baeyer-Villiger reactions. In the present review, the molecular basis for this promiscuous hydrolase capability, as well as identified applications are reviewed and described in detail. [Copyright &y& Elsevier]

Abstract: In recent years, the Carica papaya lipase (CPL) is attracting more and more interest. This hydrolase, being tightly bonded to the water-insoluble fraction of crude papain, is thus considered as a “naturally immobilized” biocatalyst. To date, several CPL applications have already been described: (i) fats and oils modification, derived from the sn-3 selectivity of CPL as well as from its preference for short-chain fatty acids; (ii) esterification and inter-esterification reactions in organic media, accepting a wide range of acids and alcohols as substrates; (iii) more recently, the asymmetric resolution of different non-steroidal anti-inflammatory drugs (NSAIDs), 2-(chlorophenoxy)propionic acids, and non-natural amino acids. Taking into account the novelty and the current interest of the topic, this review aims to highlight the origin, features, and applications of the C. papaya lipase, with the objective to prompt research groups to further investigate the spectra of applications that this emerging and versatile CPL could have in the future. [Copyright &y& Elsevier]

Abstract: Candida rugosa lipase (CRL) is one of the enzymes most frequently used in biotransformations. However, there are some irreproducibility problems inherent to this biocatalyst, attributed either to differences in lipase loading and isoenzymatic profile or to other medium-engineering effects (temperature, a w, choice of solvent, etc.). In addition, some other properties (influence of substrate and reaction conditions on the lid movement, differences in the glycosylation degree, post-translational modifications) should not be ruled out. In the present paper the recent developments published in the CRL field are overviewed, focusing on: (a) comparison of structural and biochemical data among isoenzymes (Lip1–Lip5), and their influence in the biocatalytical performance; (b) developments in fermentation technology to achieve new crude C. rugosa lipases; (c) biocatalytical reactivity of each isoenzyme, and methods for characterising them in crude CRL; (d) state-of-the-art of new applications performed with recombinant CRLs, both in CRL-second generation (wild-type recombinant enzymes), as well as in CRL-third generation, (mutants of the wt-CRL). [Copyright &y& Elsevier]

Abstract: The yeast Candida antarctica produces two different lipases, lipases A and B. While lipase B (CAL-B) is probably the mostly employed hydrolase in the biocatalysis field, the use of the lipase A (CAL-A) has been rather scarce and consequently its tridimensional structure has not been elucidated yet. However, CAL-A is a useful biocatalyst with many different applications that have been described especially in the last few years. Its attractiveness results from its unique features among hydrolases: the high thermostability, allowing operation at T >90°C; the ability to accept tertiary and sterically hindered alcohols, which has recently been attributed to the existence of a specific aminoacidic sequence in the active site; the sn-2 recognition in hydrolysis of triglycerides; the selectivity towards trans-fatty acids; the stability in the acidic pH range. Furthermore, it is considered to be an excellent biocatalyst for the asymmetric synthesis of amino acids/amino esters, due to its chemoselectivity towards amine groups. Considering all these aspects, in the present review, the origin, the properties and the applications of the CAL-A are briefly described and discussed, pointing out the unique characteristics of this biocatalyst. [Copyright &y& Elsevier]

Nitriles comprise a broad group of chemicals that are currently being industrially produced and used in fine chemicals and pharmaceuticals, as well as in bulk applications, polymer chemistry, solvents, etc. Aldoxime dehydratases catalyze the cyanide-free synthesis of nitriles starting from aldoximes under mild conditions, holding potential to become sustainable alternatives for industrial processes. Different aldoxime dehydratases accept a broad range of aldoximes with impressive high substrate loadings of up to >1 Kg L−1 and can efficiently catalyze the reaction in aqueous media as well as in non-aqueous systems, such as organic solvents and solvent-free (neat substrates). This paper provides an overview of the recent developments in this field with emphasis on strategies that may be of relevance for industry and sustainability. When possible, potential links to biorefineries and to the use of biogenic raw materials are discussed. [ABSTRACT FROM AUTHOR]

Whole-cell biocatalysis with the use of baker's yeast is demonstrated in different mixtures of water with deep eutectic solvents (DESs; choline chloride/glycerol, 1:2 mol/mol). Enantioselective ketone reduction is observed for long reaction times (>200 h), which suggests that the whole cells remain stable in these neoteric solvents. By changing the proportion of the DES added, a complete inversion of enantioselectivity is observed, from approximately 95 % enantiomeric excess ( ee) ( S) in pure water to approximately 95 % ee ( R) in the pure DES. Presumably, some ( S)-oxidoreductases present in baker's yeast are inhibited by DESs. [ABSTRACT FROM AUTHOR]

The application of biocatalysis and White Biotechnology tools in chemical areas concerning the production of bulk compounds and other related low-added value products (with high volumes) has been gaining importance in recent years. The expected drivers of biocatalysis for these sectors are energy savings, regioselectivity (leading to cleaner products), the possibility of using thermolabile substrates, as well as the generation of less by-products and manageable wastes. This paper explores some recent industrial granted patents related to biocatalysis and bulk chemicals. Several patents have been identified in fields such as biodiesel and esterification reactions, and sugar or furan chemistry. Overall, innovative strategies involve the identification of novel enzymes, the set-up of improved immobilization methods, as well as novel reactor designs that can offer improved performances and economics. The reported examples indicate that biocatalysis can certainly offer opportunities for these areas as well, far from the typical pharmaceutical and fine chemical applications often reported in the literature. [ABSTRACT FROM AUTHOR]

The article discusses a study which assessed whether the enzymatically formed acetaldehyde can be used as a further reagent for domino processes in aqueous media. The study investigated the hydrolytic activity of immobilized lipase B from Candida antarctica, CAL-B. It tested three different vinyl esters in the reaction under various conditions. It was shown that acetaldehyde can be efficiently formed in situ by means of lipases in aqueous solutions and in biphasic systems.

The article discusses a study on a novel type of enzymes, the nitrile reductases or nitrile oxidoreductases which catalyze the four-electron reduction to afford primary amines from nitriles. It cites efforts for the elucidation of the nitrile reduction mechanism proposing a reaction pathway that involve two molecules of cofactor in each catalytic turnover. It suggests the these enzymes may be widely spread in nature and opens the lead for new biocatalytic applications in the nitrile chemistry.

A non-innocent biogenic solvent? The use of Cyrene™ as solvent in biocatalysis may be advantageous. Its ability to form geminal diols and Cygnet-type structures leads to solvent gradients with tailored polarities, offering promising options for enzymatic processes (from its use in aqueous media as co-solvents, to synthetic purposes as non-conventional media). [Display omitted] • Cyrene is useful as organic solvent for lipophilizations enzymatic reactions. • The immobilized CALB is active in Cyrene, yielding up to ∼ 10 g product L−1. • CALB-CLEA remain stable in Cyrene, enabling its use along several catalytic cycles. • CALB displays hydrolytic reactions using Cyrene as cosolvent. • CALB-CLEA remain fully active over 6 cycles at 30 % v/v Cyrene. An emerging biogenic aprotic solvent is the cellulose-derived dihydrolevoglucosenone, or 6,8-dioxabicyclooctanone (Cyrene™). This paper explores the use of Cyrene in lipase-catalyzed biotransformations, both in aqueous solutions – as co-solvent – as well as non-conventional media for synthesis. Cyrene is useful as organic solvent for lipophilizations using benzoic acid and glycerol as a model system for substrates with unpaired solubilities. The immobilized lipase B from Candida antarctica is active in Cyrene, yielding up to ∼ 10 g product L−1. Interestingly, crosslinked aggregates immobilized lipases (CLEA) remain significantly stable in Cyrene, enabling its use along several catalytic cycles. Cyrene is highly hygroscopic and forms geminal-diol structures with water, leading to solvent mixtures with a (tailored) gradient of polarities, what may be promising for biocatalysis in aqueous solutions. CALB displays hydrolytic reactions with different proportions of Cyrene as cosolvent, and CLEA derivatives remain fully active over 6 cycles at 30 % v/v Cyrene. [ABSTRACT FROM AUTHOR]

[Display omitted] • Lipase from Pseudomonas stutzeri was immobilized through cross-linking aggregates. • The new derivative is active in a synthesis reaction using DES as reaction media. • The biocatalyst is stable and can be reused several times. • Cross-linking aggregates are a simple tool to obtain active biocatalysts in DES. The use of deep eutectic solvents (DES) with buffer as cosolvent (up to 10 % v/v) leads to low-viscous media in which lipases can perform synthetic reactions, instead of hydrolysis. This paper explores the immobilization of Pseudomonas stutzeri lipase (TL) in cross-linking aggregates (CLEA) to deliver robust derivatives that are active in media like choline chloride – glycerol DES with buffer as cosolvent. While the free TL enzyme was markedly inactive in these media, TL-CLEA derivatives perform esterifications and can be reused several times. Overall, results are consistent with previous experiments reported for other lipases in these DES-water media and confirm that CLEA immobilization turns out a very useful and straightforward alternative for generating active (bio)catalysts for DES-aqueous media systems. Immobilized systems open the possibility of performing continuous processes in low-viscous DES-buffer media. [ABSTRACT FROM AUTHOR]

The valorization of lignin-derived feedstocks by catalytic means enables their defunctionalization and upgrading to valuable products. However, the development of productive, safe, and low-waste processes remains challenging. This paper explores the industrial potential of a chemoenzymatic reaction performing the decarboxylation of bio-based phenolic acids in wet cyclopentyl methyl ether (CPME) by immobilized phenolic acid decarboxylase from Bacillus subtilis, followed by a base-catalyzed acylation. Key-to-success is the continuous control of water activity, which fluctuates along the reaction progress, particularly at high substrate loadings (triggered by different hydrophilicities of substrate and product). A combination of experimentation, thermodynamic equilibrium calculations, and MD simulations revealed the change in water activity which guided the integration of water reservoirs and allowed process intensification of the previously limiting enzymatic step. With this, the highly concentrated sequential two-step cascade (400 g·L–1) achieves full conversions and affords products in less than 3 h. The chemical step is versatile, accepting different acyl donors, leading to a range of industrially sound products. Importantly, the finding that water activity changes in intensified processes is an academic insight that might explain other deactivations of enzymes when used in non-conventional media. Lignin-derived phenolic acids can be upgraded to styrene derivatives through chemoenzymatic processes, however, the scale-up of such processes remains challenging. Here, the authors find that controlling the water activity during the decarboxylation of bio-based phenolic acids, including through the integration of a water reservoir, enables high conversions and efficient reaction times, that can be combined with a versatile acyl donor substrate scope. [ABSTRACT FROM AUTHOR]

• Lipase-CLEA aggregates can be efficiently immobilized in Lentikats®, leading to a highly robust biocatalyst. • The new biocatalyst can be used in low viscous non-conventional Deep Eutectic Solvent – Buffer systems. • The new biocatalyst displays outstanding stability and straightforward recovery. The immobilization of lipases in cross linked aggregates (CLEA) leads to a robust biocatalyst that remains very stable in low viscous non-conventional Deep Eutectic Solvents – Buffer mixtures. To reinforce that stability, and to facilitate the biocatalyst recovery, this paper explores the immobilization of Lipase-CLEA derivatives in Lentikats®. This double immobilization can be successfully used in esterifications in DES-buffer media, using substrates of unpaired solubilities (e.g. benzoic acid and glycerol), in batch and continuous processes, and reaching full conversion. Under these conditions, the derivatives display an improved stability (compared to the Lipase-CLEA derivatives) and enable the reuse of the reaction media in continuous devices for at least 6 cycles under non-optimized conditions, accumulating 10 g product L−1, enhancing the productivity, and opening exciting future options for sustainable chemistry. [ABSTRACT FROM AUTHOR]

The combination of Deep-eutectic-solvents (DES) with water as "co-solvent" enables a low-viscous reaction medium that keeps its "non-conventional" nature and thus enables synthetic lyophilization reactions (e.g. esterification) catalyzed by hydrolases. Substrates with different polarity may be employed. This paper shows how the enzyme immobilization with cross-linking aggregates (CLEA) leads to highly stable and active immobilized catalysts in different DES. As a remarkable case, when choline chloride-glycerol DES is used, CLEA derivatives of Candida antarctica lipase B (CLEA-CALB) are stable for at least 14 days without any loss of activity. The immobilized biocatalysts are applied in non-viscous DES-water blends (8% v/v) to catalyze the esterification of benzoic acid and glycerol to furnish glyceryl monobenzoate (α-MBG) in productivities of ∼35 g α-MBG L−1d−1. Compared to other commercial immobilized CALB, the CLEA-CALB derivatives rendered more product (higher conversions by 30%). Moreover, CLEA derivatives were successfully reused for six times without any loss of activity. Given the ease of immobilization (CLEA), their excellent performance in DES and the low viscosity of the DES-water blends, the reported approach may be useful for many synthetic procedures and even for continuous processes with largely optimized outcomes. [ABSTRACT FROM AUTHOR]

Highlights • Low viscous DES-water blends can be used in continuous processes for biocatalysis. • The pH control increases the conversion, using DES in a fed-batch reactor. • Fed-batch reactor reached the highest concentration of product 33 g/L. • The semicontinuous and continuous bioreactors reached similar productivities. • Biocatalyst stability was high in a packed-bed reactor, without loss for 10 days. Abstract This work explores for the first time the use of Deep Eutectic Solvents (DES) with phosphate buffer 100 mM pH 7 as cosolvent (10% v/v) in biocatalytic reactions in fed-batch and packed-bed bioreactors. The lipase-catalyzed esterification of glycerol and benzoic acid is studied, as it involves two substrates with different polarities (for which DES are needed). In the fed-batch bioreactor, the highest conversion (90%) was obtained at a substrate flow rate of 0.01 mL/min. The fed-batch operation increased the conversion by 59% compared to the batch mode. Regarding productivity, semi-continuous and continuous bioreactors showed analogous results. Upon recirculation of the reaction media in the continuous bioreactor, a conversion of 67% was achieved in 7 cycles of operation. The stability of the biocatalyst in the packed-bed bioreactor decreased only 2% in 10 days, demonstrating the attractiveness that low viscous DES-water mixtures with continuous processes may have. [ABSTRACT FROM AUTHOR]

Lignin is the major aromatic biopolymer in nature, and it is considered a valuable feedstock for the future supply of aromatics. Hence, its valorisation in biorefineries is of high importance, and various chemical and enzymatic approaches for lignin depolymerisation have been reported. Among the enzymes known to act on lignin, β-etherases offer the possibility for a selective cleavage of the β-O-4 aryl ether bonds present in lignin. These enzymes, together with glutathione lyases, catalyse a reductive, glutathione-dependent ether bond cleavage displaying high stereospecificity. β-Etherases and glutathione lyases both belong to the superfamily of glutathione transferases, and several structures have been solved recently. Additionally, different approaches for their application in lignin valorisation have been reported in the last years. This review gives an overview on the current knowledge on β-etherases and glutathione lyases, their biochemical and structural features, and critically discusses their potential for application in biorefineries. [ABSTRACT FROM AUTHOR]

Glutathione-dependent β-etherases and glutathione lyases are key-enzymes for the biocatalytic depolymerization of lignin. In the first step, the nucleophilic attack of glutathione to the common β-O-4-aryl-ether motif in lignin is catalyzed by β-etherases and afterwards the glutathione is removed again by the action of glutathione lyases. Given their potential impact for lignin valorization, in this paper novel glutathione lyases are reported and biocatalytically characterized based on lignin model compounds. As a result, an enzyme exhibiting increased thermostability and lowered enantioselectivity – key features for implementation of glutathione lyases in enzymatic lignin depolymerization processes – was identified. Furthermore, first mutational studies of these enzymes revealed the possibility to further alter the activity as well as enantioselectivity of glutathione lyases by means of protein engineering. From a practical perspective, one-pot multi-step processes combining β-etherases and glutathione lyases are successfully set-up, giving hints on the potential that the implementation of these biocatalysts may bring for biorefinery purposes. [ABSTRACT FROM AUTHOR]

Highlights: [•] Chitosan-aerogels catalyze biodiesel-type transesterifications. [•] Chitosan is presently a waste that can be valorized. [•] Other polysaccharides (e.g. alginic acid) may be used as catalysts as well. [Copyright &y& Elsevier]

Deep eutectic solvents (DESs) are formed by mixing quaternary ammonium salts (e.g., choline chloride) and hydrogen-bond donors (e.g., glycerol or urea), which leads to biodegradable and readily available ionic solvents at room temperature. Analogous to other ionic liquids, DESs represent a promising reaction media if hydrophobic and hydrophilic substrates need to be combined. This paper assesses DESs as reaction media for chymotrypsin-catalyzed peptide synthesis. After careful determination of the reaction conditions (e.g., water content, enzyme loading), α-chymotrypsin displayed high activity for peptide synthesis in choline chloride/glycerol mixtures to afford productivities of ca. 20 g L-1 h-1 and with complete selectivity for the peptide, which is in contrast to the detrimental hydrolysis pathway observed in aqueous media. The nonimmobilized suspended enzyme could be reused several times by simple filtration with excellent to moderate activities. Overall, the results reported suggest that choline chloride based DESs may become promising neoteric solvents for peptide synthesis through biocatalysis. [ABSTRACT FROM AUTHOR]

For a knowledge-based design of enzyme catalysis in deep eutectic solvents (DESs), the influence of the DES properties (e.g., water activity and viscosity) and the impact of DESs and their individual components must be assessed. This paper investigates three different DESs: choline chloride–glycerol (ChCl–Gly), choline chloride–ethylene glycol (ChCl–EG), and ethyl ammonium chloride–glycerol (EACl–Gly). The specific activity and half-life time of horse liver alcohol dehydrogenase (HLADH) were experimentally determined in these DESs with water contents ranging from 0%–100%. HLADH showed limited activity in neat DESs, which was enhanced by adding water. Experiments with individual DES components of ChCl–Gly were carried out to clarify their individual influence. Glycerol acts as a strong stabilizer for the enzyme, whereas choline chloride's results are deleterious. To understand the experimental findings, molecular dynamics (MD) simulations were carried out to quantify the solvation layer and calculate the spatial distribution of the solvent molecules around HLADH. The experimental and the in silico approach suggests that designing novel DESs with higher glycerol loadings would result in improved media for biocatalysis. This is demonstrated by performing the reduction of cinnamaldehyde to cinnamyl alcohol—a relevant compound for the food industry and cosmetics—in ChCl–Gly (1 : 9 molar ratio), with 20 vol% water to decrease the viscosity. HLADH is highly active and stable under these new conditions, giving promising productivity (15.3 g L−1 d−1). This paper demonstrates that DESs can be designed to be both substrate-solubilizers and enzyme-compatible, opening new research lines for green chemistry and biocatalysis. [ABSTRACT FROM AUTHOR]

Enzymatic multistep reactions are presently an important research field, from which integrated and efficient synthetic protocols can be created, accompanied by a diminished waste formation (avoiding downstream units operations). This article explores the benzaldehyde lyase (BAL) catalyzed crossed carboligation of benzaldehyde with different aliphatic aldehydes to afford optically active α-hydroxyketones. To this end, different biobased aliphatic alcohols were in situ oxidized to aldehydes by oxidase from Hansenula sp. and subsequently carboligated with benzaldehyde by BAL in the same reactor system. For short nonbranched aliphatic alcohols, moderate to high conversions in carboligations (15-99 %) with excellent enantioselectivities (98-99 %, R), were achieved. Both enzymes also exhibited activities at high concentrations of benzaldehyde (up to 200 m M) and with butanol as cosolvent, albeit at the cost of lower conversions, presumably owing to kinetic reasons. After needed optimization of the biocatalyst (e.g., through genetic evolution, whole-cell setup) and the process setup (e.g., stepwise addition of substrates, reaction time), the herein reported concept might provide promising entries in the field of asymmetric synthesis, delivering useful building blocks starting from biobased materials, and in an integrative manner. [ABSTRACT FROM AUTHOR]

Abstract: Deep-Eutectic-Solvents (DES) were assessed for the separation of alcohol–ester mixtures, for example those typically obtained from catalytic kinetic resolutions of racemic alcohols. DES can efficiently dissolve molecules containing hydrogen-bond-donors (alcohols), whereas esters remain as second phase. By using this concept, tedious separation chromatographic steps may be easily overcome with bio-based non-hazardous solvents. [Copyright &y& Elsevier]

Upon CO2 pressurization, 2-methyltetrahydrofuran (2-MTHF) expands. The expanded phase exerts different solvation properties, triggering fractional lignin precipitation. Depending on the actual CO2 pressure, fractions of lignin with a gradient of different sizes and chemical properties are selectively precipitated. A range of raw materials and applications may be thus reached. [ABSTRACT FROM AUTHOR]

In nature, enzymes conventionally operate under aqueous conditions. Because of this, aqueous buffers are often the choice for reaction media when enzymes are applied in chemical synthesis. However, to meet the demands of an industrial application, due to the poor water solubility of many industrially relevant compounds, an aqueous reaction system will often not be able to provide sufficient substrate loadings. A switch to a non-aqueous solvent system can provide a solution, which is already common for lipases, but more challenging for biocatalysts from other enzyme classes. The choices in solvent types and systems, however, can be overwhelming. Furthermore, some engineering of the protein structure of biocatalyst formulation is required. In this review, a guide for those working with biocatalysts, who look for a way to increase their reaction productivity, is presented. Examples reported clearly show that bulk water is not necessarily required for biocatalytic reactions and that clever solvent systems design can support increased product concentrations thereby decreasing waste formation. Additionally, under these conditions, enzymes can also be combined in cascades with other, water-sensitive, chemical catalysts. Finally, we show that the application of non-aqueous solvents in biocatalysis can actually lead to more sustainable processes. At the hand of flowcharts, following simple questions, one can quickly find what solvent systems are viable. [ABSTRACT FROM AUTHOR]

Abstract: Silica-supported piperazine was found to be an efficient catalyst for aldol reactions of aromatic aldehydes and ketones with straightforward product isolation and catalyst reuse. Furthermore, the catalyst is active in Knoevenagel-type reactions to afford coumarin derivatives, using 2-methyltetrahydrofuran (2-MeTHF) as a novel bio-based solvent. [Copyright &y& Elsevier]

A successful biorefinery needs to align suitable pretreatment with sustainable production of biomasses. Herein, four perennial plants, (Sida, Silphium, Miscanthus and Szarvasi) regarded as promising feedstocks for biorefineries were subjected to the OrganoCat pretreatment. The technology was successfully applied to the different perennial plants revealing that pretreatment of grasses was more efficient than of non-grasses. Thorough analyses of the lignocellulose – before and after fractionation – enabled a detailed description of the fate of cellulosic, non-cellulosic polysaccharides and lignin during the pretreatment. Especially Szarvasi pulp displayed outstanding results in terms of fractionation efficiency and enzymatic digestibility, though in all cases successful lignocellulose fractionation was observed. These insights into the structural composition of different perennial plant species and the impact of the OrganoCat pretreatment on the plant material leads to useful information to strategically adapt such processes to the individual lignocellulosic material aiming for a full valorisation. [ABSTRACT FROM AUTHOR]

Lignin is a biomass-derived aromatic polymer that has been identified as a potential renewable source of aromatic chemicals and other valuable compounds. The valorization of lignin, however, represents a great challenge due to its high inherent functionalization, what compromises the identification of chemical routes for its selective depolymerization. In this work, an in vitro biocatalytic depolymerization process is presented, that was applied to lignin samples obtained from beech wood through OrganoCat pretreatment, resulting in a mixture of lignin-derived aromatic monomers. The reported biocracking route comprises first a laccase-mediator system to specifically oxidize the Cα hydroxyl group in the β- O-4 structure of lignin. Subsequently, selective β- O-4 ether cleavage of the oxidized β- O-4 linkages is achieved with β-etherases and a glutathione lyase. The combined enzymatic approach yielded an oily fraction of low-molecular-mass aromatic compounds, comprising coniferylaldehyde and other guaiacyl and syringyl units, as well as some larger (soluble) fractions. Upon further optimization, the reported biocatalytic route may open a valuable approach for lignin processing and valorization under mild reaction conditions. [ABSTRACT FROM AUTHOR]

Deep eutectic solvents (DES) have emerged as promising solutions in many areas of chemistry. DES have many of the advantages of ionic liquids (e.g., low vapor pressure, nonflammability, tunability, etc.) but have lower costs, straightforward syntheses, and are often biodegradable. In this minireview, we discuss briefly the applications of DES in areas such as biotransformations, organocatalysis, and combinations of these that lead to multistep processes. DES can be used as solvents, cosolvents, performance additives for bio- and organocatalysis, tools to anchor organocatalysts selectively, or for extraction/separation. Several DES have been used both as solvents and organocatalysts at the same time. Given their promising features, it may be expected that many applications in these areas will appear in the coming years. [ABSTRACT FROM AUTHOR]

Deep-eutectic solvents (DES) are cost-effective, nonhazardous solvents that may be used in biocatalysis as nonconventional media that enable biotransformations with industrially sound high substrate loadings. Based on promising prognoses, this paper explores successfully the use of whole cells overexpressing oxidoreductases as biocatalysts for the stereoselective reduction of ketones in different DES-aqueous-media solutions. Enzymes like Ralstonia sp. alcohol dehydrogenase (ADH) and horse liver ADH, overexpressed in Escherichia coli, remain active at different DES proportions (for example, DES:buffer, 80:20 v/v) to enable nonconventional biotransformations. Furthermore, the enantiomeric excesses obtained for a broad range of aromatic substrates increase significantly if DES is added, which provides a useful tool for combining high substrate loadings and improved enzymatic selectivities while working with nonhazardous solvents. Different DES are successfully employed, which gives hints of the potential that these emerging solvents may have. [ABSTRACT FROM AUTHOR]

The fractionation of lignocellulose in its three main components, hemicellulose, lignin and cellulose pulp can be achieved in a biphasic system comprising water and bio-based 2-methyltetrahydrofuran (2-MeTHF) as solvents and oxalic acid as catalyst at mild temperatures (up to 140 °C). This so-called OrganoCat concept relies on selective hemicellulose depolymerization to form an aqueous stream of the corresponding carbohydrates, whereas solid cellulose pulp remains suspended and the disentangled lignin is to a large extent extracted in situ with the organic phase. In the present paper, it is demonstrated that biomass loadings of 100 g L−1 can be efficiently fractionated within 3 h whereby the mild conditions assure that no significant amounts of by-products (e.g. furans) are formed. Removing the solid pulp by filtration allows to re-use the water and organic phase without product separation in repetitive batch mode. In this way, (at least) 400 g L−1 biomass can be processed in 4 cycles, leading to greatly improved biomass-to-catalyst and biomass-to-solvent ratios. Economic analysis of the process reveals that the improved biomass loading significantly reduces capital and energy costs in the solvent recycle, indicating the importance of process integration for potential implementation. The procedure was successfully scaled-up from the screening on bench scale to 3 L reactor. The feedstock flexibility was assessed for biomasses containing moderate-to-high hemicellulose content. [ABSTRACT FROM AUTHOR]

Benzaldehyde lyase (BAL) results in an efficient biocatalyst for the umpolung carboligation of furfural, HMF, and mixtures of them, leading to blended C10–C12 platform chemicals. Subsequently, the mixing and gentle heating (＜100 °C) of the formed hydroxy-ketone with choline chloride leads to the formation of a novel biomass-derived deep-eutectic-solvent. [ABSTRACT FROM AUTHOR]

Highlights: [•] Enzymatic screening to identify the potential application of biocatalysis to therapeutically relevant substrates: tetrahydropyrazolo[1,5-α]pyrimidines. [•] CAL-B-catalyzed enantioselective biotransformations: kinetic resolution of chiral substrates with asymmetric centres located far from the catalytic centre. [•] Proof of concept for process optimization: screening of bio-based co-solvents. [ABSTRACT FROM AUTHOR]

Hydrolases catalyze synthetic reactions in nonaqueous media, whereas they perform hydrolysis under aqueous solutions. An acyl transferase from Mycobacterium smegmatis (MsAcT) is able to catalyze synthetic reactions in buffer because of its highly hydrophobic active site, which enables efficient transesterification reactions even at 99.9 % v/v buffer solution. This unique feature of MsAcT among hydrolases may open new opportunities to conduct synthetic (bio)catalysis in aqueous media. With these goals in mind, this paper explores some evidence of such potential: MsAcT can perform enantioselective transesterifications (e.g., ( S)-2-octanol), which could be combined with other aqueous multistep (asymmetric) reactions; 5-hydroxymethylfurfural (HMF) can be esterified to produce more hydrophobic and easily extractable HMF esters (e.g., for downstream processing or wastewater treatment); and upon addition of dilute H2O2, MsAcT works efficiently as a perhydrolase to form in situ peracids-in bulk water-that can be used for oxidations (e.g., furfural to furoic acid oxidation). Overall, these and many other new applications can be envisaged by using MsAcT in aqueous solutions. [ABSTRACT FROM AUTHOR]

Abstract: Organocatalysis is assessed for the first time in the synthesis of purine and pyrimidine acyclic nucleosides providing high yields and straightforward work-up procedures. Nucleobases containing aldehydes are catalytically ligated (C–C bond formation) to acetone or to phosphonate-containing ketones by means of pyrrolidine or silica-immobilized piperazine as amine-based organocatalysts. [Copyright &y& Elsevier]

Abstract: Aldol condensations between sugar-derived dehydrated aldehydes (e.g. furfural) and acetone have been proposed as a route to provide useful biomass-derived chemicals. In the quest of sustainable catalytic ways for such aldol condensations, this paper assesses the use of dried chitosan-gels as naturally-immobilized, readily available and non-hazardous amino-based organocatalysts. At room temperature chitosan dried gels are not suitable catalysts for the desired reaction. However, at higher temperatures (>90°C) reaction proceeds efficiently either in solvent-free systems (with addition of catalytic amounts of water) or in water. The set-up of closed reactor set-ups (thermoshakers or microwave reactions) proved highly beneficial for the reaction outcome. Furthermore, chitosan dried gels were successfully re-used for a number of cycles. An efficient catalyst drying method (either lyophilization or scCO2 drying) was crucial to achieve virtually full conversions in 4h. After pertinent further process optimization, dried chitosan-gels may become very useful catalysts for their use in biomass-based reactions in biorefineries. [Copyright &y& Elsevier]

Cellulases are promising catalysts for the depolymerization of cellulose under mild conditions. Reengineered cellulases are required to match application demands in biorefineries and to avoid cost-intensive downstream processing. This manuscript provides a novel fluorescence-based high throughput screening method for directed evolution of cellulases, based on 4-methylumbelliferyl-β-d-cellobioside (4-MUC). The 4-MUC high throughput screening system was successfully employed to identify CelA2 variants with enhanced stability and activity in mixtures of water with deep eutectic solvents like choline chloride : glycerol (ChCl : Gly), and seawater. The cellulase variant 4D1 (L21P; L184Q; H288R; K299I; D330G; N442D) was isolated and showed, compared to wild type, an increase in specific activity in 30% (v/v) ChCl : Gly (7.5-fold; 0.4 to 3.0 U mg−1) and in concentrated seawater (1.6-fold; 5.5 to 9.3 U mg−1). In addition, the residual activity of 4D1 in the presence of 3-fold concentrated seawater is unaffected whereas CelA2 wild type loses ＞50% of its activity. Furthermore, the position H288 was identified as a key position for activity and resistance in 4D1. [ABSTRACT FROM AUTHOR]

A one-pot multi-step approach comprising enzymatic oxidation-hydroxymethylation-reduction enables the synthesis of optically active α-aryl vicinal diols with high yields and enantioselectivities. Formaldehyde required for the hydroxymethylation step is enzymatically produced in situ using less hazardous methanol as substrate. [ABSTRACT FROM AUTHOR]